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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Dec 2018 to 16 Dec 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Justification for study design:
ECHA requirement - Screening for reproductive/developmental toxicity (Annex VIII, Section 8.7.1.; OECD 421 or 422) in rats, oral route.

Justification of dose selection rationale::

Doses were selected from a 14-day Dose-Ranger Finding study to determine the preliminary effects of Bourgeonal following daily exposure for 14 consecutive days to male and female rats, and to provide information for selection of dose levels to be used in subsequent OECD 422 study.

A total of 40 Crl:CD(SD) Sprague Dawley P generation rats (20 rats/sex) were randomly assigned to dose groups, 5 rats/sex/group. Formulations of the test substance, Bourgeonal, or the control substance, Corn Oil, were administered orally by gavage three times (approximately 6 hours apart) daily for 14 consecutive days at 0 (Control), 5, 25, and 50 mg/kg/day.

Study parameters included: viability, clinical observations, body weights and body weight changes, food consumption, urinalysis (acid metabolites), macroscopic and microscopic observations, organ weights, and sperm evaluations (motility and concentration).

There were no Bourgeonal-related mortalities in females at any dose or males at 5 and 25 mg/kg/day. There were two mortalities at 50 mg/kg/day in males. One male was found dead on SD 14. Although there were no clinical signs prior to death or macroscopic findings during necropsy examination the likelihood of Bourgeonal-related toxicity cannot be eliminated. The other male was euthanized on SD 1 due to adverse clinical condition. This early death was considered to be unrelated to Bourgeonal because the death was attributed to the jugular blood collection procedure based on the macroscopic findings and timing of death. All other animals survived to scheduled euthanasia on SD 15.

Bourgeonal-related clinical signs were limited to females in the 25 and 50 mg/kg/day dose groups, and included suspected dehydration (based on skin turgor) and a low incidence of hunched posture and thin appearance. There were no Bourgeonal-related clinical observations in males at any dose.

In females, mean body weight losses of -15.4 g and -33.0 g were observed at 25 and 50 mg/kg/day, respec-tively, compared to a mean body weight gain of +13.3 g in controls for the interval of SD 1 to 15. In addition, lower mean body weights were observed in females on SD 7 and SD 15 at 25 mg/kg/day (91% to 96% of control) and on SD 3 and SD 15 at 50 mg/kg/day (82% to 95% of control). There were no Bourgeonal-related effects on mean body weights or mean body weight gain in males at any dose.

Lower mean food consumption was observed in females at 25 mg/kg/day for the intervals of SD 1 to 5, SD 5 to 8, and SD 12 to 14 (84% to 94% of control) and at all tabulated intervals between SD 1 and SD 14 and overall for the interval of SD 1 to 14 at 50 mg/kg/day (36% to 83% of control). There were no Bourgeonal-related effects on mean absolute food consumption in males at any dose.

At necropsy examination, Bourgeonal-related macroscopic findings were limited to decreased testicular size which was observed in one male at 50 mg/kg/day. In addition, Bourgeonal-related differences in mean absolute and relative organ weights were limited to the testes (50 mg/kg/day) and liver (≥5 mg/kg/day) in the males and the ovaries (≥25 mg/kg/day) and uterus (50 mg/kg/day) in females. Of note: due to prominently decreased food consumption and weight loss indicative of sustained negative energy balance in females, the relationship of uterine and ovarian findings cannot be definitively determined (direct test substance effect, vs. secondary to negative energy balance) due to the impact of negative energy balance on reproductive structures that are non-essential for survival. With the exception of the decreased ovarian weights, each of the changes in the testes, liver, and uterus had a histologic correlate of hypertrophy or atrophy/degeneration.

In males, Bourgeonal-related microscopic findings were observed in the testes at ≥5 mg/kg/day (vacuolation and degeneration of seminiferous tubular epithelium, Sertoli cell vacuolation) with secondary effects in the epididymides at ≥25 mg/kg/day (cribriform change, cellular debris, and hypospermia). The vacuolation noted in the seminiferous tubule was characterized by fine microvesicular vacuolation within the cytoplasm of seminiferous tubule epithelium and uniformly affected all stages of spermatogenesis (spermatogonia, spermatocyte, and spermatid). In females, Bourgeonal-related microscopic findings were observed in the uterus at 50
mg/kg/day (uterine atrophy). Bourgeonal-related microscopic findings were also observed in the liver of both males and females, including periportal to midzonal hepatocellular vacuolation at ≥5 mg/kg/day and centrilobular hepatocellular hypertrophy in males at ≥25 mg/kg/day and in females at 50 mg/kg/day.

Bourgeonal-related effects in sperm motility were observed at all doses, including reductions in sperm motility at 5 mg/kg/day (77% vs. 84% in controls) and little to no sperm at ≥25 mg/kg/day (18% and 3%, respectively, vs. 84% in controls). All sperm samples that were analyzed at ≥25 mg/kg/day contained headless and detached sperm, with the exception of one sperm sample at 25 mg/kg/day. The infrequent increased spermatid head retention by the Sertoli cells, degeneration of maturing spermatids, round spermatids, and/or elongating spermatids, exfoliation/degeneration of germ cells, increased cellular debris, and moderate to marked hypospermia that was observed microscopically in the seminiferous tubules or epididymides may have contributed to the overall decrease in sperm motility.

On SD 1, Bourgeonal acid and 4-tBBA were below the limit of detection in all male and female predose plasma samples and from males and females administered 5 mg/kg/day. Mean Bourgeonal acid concentrations were below the level of detection or generally lower than 4-tBBA concentrations from 25 and 50 mg/kg/day samples and tended to be higher in females. Mean 4-tBBA concentrations were slightly higher in females at 25 mg/kg/day but were much similar to males at 50 mg/kg/day.

On SD 14, 4-tBBA plasma concentrations quickly increased and maintained steady state from 0.5 to 24 hr in males and females at all dose levels. At doses ≥ 25 mg/kg/day, 4-tBBA concentrations in females were almost twice that of males and increased in a nearly dose proportional manner. Mean Bourgeonal acid concentrations were below the limit of detection or ≤ 1.6 μM in males and females at 5 mg/kg/day. Overall, mean Bourgeonal acid concentrations at SD 14 were 1.5 to 6x and 9 to 15x lower at 25 and 50 mg/kg/day, respectively, then concentrations observed on SD 1.

Based on these results, the no-observed-adverse-effect level (NOAEL) for Bourgeonal could not be estab-lished in the 14-day DRF study. Therefore, the maximum tolerated dose was determined to be 5mg/kg/d, for both males and females based on the adverse effects observed at this dose and with consideration to the significantly longer exposure time for animals in the OECD 422 study (orally by gavage once daily with P generation male rats actual 42 to 45 doses and P generation female rats actual 38 to 56 doses).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Bourgeonal
- Physical state: liquid
- Analytical purity: 99.0%
- Lot/batch No.: VE00121957
- Expiration date of the lot/batch:October 15, 2011

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Crl:CD(SD) strain was used because 1) it is one mammalian species accepted for use in toxicity studies and it has been widely used throughout industry; 2) this species and strain has been demonstrated to be sensitive to reproductive and developmental toxicants; and 3) historical data and experience exist at the Testing Facility.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Charles River Laboratories, Inc., Raleigh, NC,
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
29 to 35 days
- Weight at study initiation:
191-230g for males, 283-329g for females
- Housing:
Upon arrival, P generation rats were co-housed in solid-bottomed cages (2/sex/cage), except during the cohabitation period and postpartum periods (see Appendix 2, Deviations). During cohabitation, each pair of P generation rats was housed in the male rat’s nesting box (1:1). P generation females with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at Gestation Day (GD) 0 and assigned to individual housing in nesting boxes. Each P generation dam and delivered litter were housed in a common nesting box during the postpartum period, except during motor activity and functional observational battery testing. Following cohabitation, P generation males were returned to the previous premating cage mate and were monitored for incompatibility issues post-cohabitation.
Controls were housed on a separate rack from test substance treated rats (see Appendix 2, Deviations).
- Diet (e.g. ad libitum):
Rats were given Certified Rodent Diet® #5002 pelleted food (PMI® Nutrition International) available ad libitum from individual feeders throughout the study, except during designated procedures
- Water (e.g. ad libitum):
ad libitum from individual bottles attached to the nesting boxes

DETAILS OF FOOD AND WATER QUALITY:
The food was analyzed for environmental contaminants and results of the analysis are on file at the Testing Facility. There were no known contaminants in the food that would interfere with the objectives of the study.
Periodic analysis of the water was performed, and results of these analyses are on file at the Testing Facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
19-23°C
- Humidity (%):
40-70%
- Photoperiod (hrs dark / hrs light):
12h/12h

IN-LIFE DATES:
Study Initiation Date: 27 Dec 2018
Initiation of Dosing: 15 Jan 2019
Completion of In-life: 12 Mar 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The control substance, Corn Oil, NF, was aliquoted once weekly for administration to Group 1, stored at room temperature until use, and dispensed as necessary. The control substance, Corn oil, NF to be used as the vehicle to prepare the test substance dose formulations had an assigned 7 day use period. Therefore, the Corn Oil, NF parent container was used for preparation and or/aliquoted in single use aliquots for up to 7 days from the initial date of opening of the parent container. The single use aliquots were used for the duration of the study, up to the date of expiration.

The test substance, Bourgeonal, was formulated in the control substance and prepared at the appropriate concentrations. The prepared test substance dose formulation was prepared once daily, and maintained at ambient conditions, protected from light throughout the duration of use for dose administration and/or sampling. The test substance dose formulation was fully used within 4 hours of preparation. The dosing formulations were stirred continuously for at least 30 minutes prior to dosing, throughout dose administration and post-dose sampling.

P generation male rats were administered the test or control substance formulations once daily via oral gavage beginning 14 days before cohabitation with treated females, during cohabitation and continuing through the day prior to scheduled euthanasia. P generation males were exposed to the test or control substance for 42 to 45 days.
P generation female rats were administered the test or control substance formulations once daily via oral gavage beginning 14 days before cohabitation with treated males and continuing through Lactation Day (LD) 12 (rats that deliver a litter) or GD 25 (rats that did not deliver a litter).
Doses were adjusted based on the most recently recorded body weight. The gavage needle was wiped clean prior to dose administration for each rat.

F1 generation pups were not directly exposed to the test or control substance, but may have been possibly exposed during maternal gestation (in utero exposure), via maternal milk during the lactation period, or from exposure to maternal urine/feces.

The doses were selected from a 14-day Dose-Range Finding (DRF) study (CRL 20153551). Based on this study the maximum concentration dose was determined to be 5 mg/kg/dose.

Animals received the test material or vehicle control formulations orally at a volume-dosage of 15 ml/kg/dose.
Details on mating procedure:
During cohabitation, each pair of rats was housed in the male rat’s nesting box (1:1). P generation females with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ was considered to be at GD 0 and assigned to individual housing in nesting boxes. Each dam and delivered litter were housed in a common nesting box during the postpartum period, except during motor activity and functional observational battery testing. Following cohabitation, P
generation males were returned to the previous premating cage mate and were monitored for incompatibility issues post-cohabitation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate (1 mL, aliquot weights to be measured to at least 0.001 g) sets of top, middle, and bottom test substance samples for the sampling time points were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 20% of theoretical concentration. For homogeneity, the criteria for acceptability were a relative standard deviation (RSD) of concentrations of ≤ 10% for each group.
There was no Bourgeonal detected in the control samples for all pre- and postdose first, mid study, and last preparation samples.
Duration of treatment / exposure:
Bourgeonal, or the control substance, corn oil, were administered orally by gavage through mating and continuing for at least 28 days (P generation male rats) or through parturition until Day 12 of lactation (P generation female rats) at 0 (Control), 0.5, 1, and 5 mg/kg/dose.
Frequency of treatment:
Once daily beginning before cohabitation.
Details on study schedule:
Study Initiation Date: 27 Dec 2018
Initiation of Dosing: 15 Jan 2019
Completion of In-life: 12 Mar 2019
Experimental Start Date (OECD): 27 Dec 2019
Experimental Start Date (EPA): 15 Jan 2019
Experimental Completion Date (OECD): 09 Dec 2019
Experimental Termination Date (EPA): 09 Dec 2019
Doses / concentrationsopen allclose all
Dose / conc.:
0.5 mg/kg bw/day (nominal)
Dose / conc.:
1 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
P generation male rats were administered the test or control substance formulations once daily via oral gavage beginning 14 days before cohabitation with treated females, during cohabitation and continuing through the day prior to scheduled euthanasia. P generation males were exposed to the test or control substance for 42 to 45 days. P generation female rats were administered the test or control substance formulations once daily via oral gavage beginning 14 days before cohabitation with treated males and continuing through Lactation Day (LD) 12 (rats that deliver a litter) or GD 25 (rats that did not deliver a litter).

F1 generation pups were not directly exposed to the test or control substance, but may have been possibly exposed during maternal gestation (in utero exposure), via maternal milk during the lactation period, or from exposure to maternal urine/feces.
Doses were adjusted based on the most recently recorded body weight. The gavage needle was wiped clean prior to dose administration for each rat.


The in-life procedures, observations, and measurements listed below were performed for all P generation rats:
The rats were assessed for viability at least twice daily during the study.

General Appearance:
The P generation rats were observed for general appearance at least once during the acclimation period, at least once weekly during the predose estrous evaluation (females), daily during the exposure period, and on the day of scheduled euthanasia.

Detailed Clinical observations:
The P generation rats were observed for detailed clinical observations once prior to initiation of exposure (baseline), and once weekly thereafter. During the weeks of Functional Observational Battery (FOB) testing, detailed clinical observations were not
conducted on the 5 rats/sex/group.

Postdose Observations:
Postdose observations were recorded between 1 and 2 hours following each daily dose administration.

Body Weights:
P generation male rats had body weights recorded on the day after arrival at the Testing Facility, on the first day of exposure, at least once weekly thereafter, and on the day of scheduled euthanasia.
P generation female rats had body weights recorded on the day after arrival at the Testing Facility, on the first day of exposure, at least once weekly thereafter, and on GD 0, 4, 7, 11, 14, 17, 20, and 25 (for P generation females with no confirmed mating date), and on LD 0, 3, 6, 9, and 12. A terminal weight was also recorded.

Food Consumption:
In P generation males, food consumption was recorded at least once weekly during the exposure period and once during the week of euthanasia (food left value). In P generation females, food consumption was recorded at least once weekly during the exposure period, on GD 0, 4, 7, 11, 14, 17, 20, and 25 (for P generation females with no confirmed mating date) and on LD 0, 3, 6, 9, and 12.

Estrous Cycle Evaluations:
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Samples were collected from P generation females beginning 13 days prior to treatment, the first 14 days of treatment and during cohabitation, and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period. Also, on the day of scheduled euthanasia (LD 13), an examination of vaginal cytology was performed prior to necropsy examination to determine the stage of estrous cycle.

Cohabitation:
P generation rats were assigned to cohabitation (i.e., pairing), one male per one female (within each dose group). The cohabitation period consisted of a maximum of 14 days. P generation females that did not mate with a P generation male within the first 7 days of cohabitation were assigned an alternate P generation male (same dose group) that successfully mated with a P generation female from the same dose group and remained in cohabitation for a maximum of 7 additional days. P generation females observed with spermatozoa in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be GD 0 and assigned to individual housing. P generation female 1420 (Group 1, 0 mg/kg/dose) did not mate after completion of the 14-day cohabitation period and was considered to be GD 0 on the last day of cohabitation. This female was assigned to individual housing (solid bottom cage) and euthanized at the discretion of the Study Director.

Natural Delivery Observations:
P generation females were evaluated for adverse clinical signs, the duration of gestation (GD 0 to the time the first pup was observed), litter size (defined by all pups delivered), and pup viability at birth.

Maternal Observations:
Maternal observations were recorded daily during the postpartum period.

Functional Observational Battery (FOB):
A functional observational battery (FOB) evaluation was conducted on one occasion for 5 rats/sex/group, during the exposure period, when possible. Due to early deaths, the evaluations were conducted on 4 female rats in Groups 1, 2, and 3 to ensure
sufficient data for clinical pathology analysis. The FOB 3,4,5,6 was conducted by an observer who was unaware of the group assignment of the rat. The observer examined the rat in its home cage, while handling the animal, and/or in an open field to assess parameters including, but not limited to the following: lacrimation, salivation, palpebral closure, prominence of the eye, pupillary reaction to light, piloerection, respiration, and urination and defecation (autonomic functions); sensorimotor responses to visual, acoustic, tactile and painful stimuli (reactivity and sensitivity); reactions to handling and behavior in the open field (excitability); gait pattern in the open field, severity of gait abnormalities, air righting reaction, visual placing response, and landing foot splay (gait and sensorimotor coordination); forelimb and hindlimb grip strength; and abnormal clinical signs including but not limited to convulsions, tremors and other unusual behavior, hypotonia or hypertonia, emaciation, dehydration, unkempt appearance and deposits around the eyes, nose, or mouth. Body temperature was measured at the completion of the FOB.

Motor Activity Evaluation
Motor activity was conducted on one occasion during the exposure period using the same five rats/sex/group that were selected for FOB evaluation, when possible.
The rats were placed in an individual enclosure held within a Smartframe containing 7 x 15 photobeams utilizing infra-red pyroelectric detectors. Movement was detected in 2 dimensions anywhere in the enclosure and was differentiated into fine movement and ambulation. Each animal was monitored for one session of 60 minutes. For the purpose of data tabulation, activity data files were reduced to Excel® format into successive periods of 10 minutes each at the completion of testing. Fine movements and ambulation were analyzed in these six 10-minute periods and compared across the dose groups.

In-life Procedures, Observations, and Measurements - F1 Generation
Preweaning
The in-life procedures, observations, and measurements listed below were performed for all F1 generation litters, with the litter as the unit of measure.

Viability Checks
Litters were observed for dead pups at least twice daily and the pups in each litter were counted once daily during the preweaning period.

Clinical Observations
Litters were observed at least once daily.

Body Weights
F1 generation pups had body weights recorded on Day 0 (birth), 3, 6, 9, and 12 postpartum.

Anogenital Distance
On Day 0 (birth) anogenital distance was recorded for all F1 generation pups using a calibrated stereomicroscope, micrometer, and ruler. The anogenital distance was measured from the cranial edge of the anus, which comes to a point, to the base of the
genital tubercle.

Nipple Retention
On Day 12 postpartum (LD 12) nipple presence was evaluated and the number present was recorded for all F1 generation male pups.

Laboratory Evaluations
Clinical Pathology:
Blood samples were collected under isoflurane/oxygen anesthesia from the inferior vena cava from 5 rats/sex/group.
Hematology Parameters:Red blood cell count; Hemoglobin concentration; Hematocrit; Mean corpuscular volume; Red blood cell distribution width; Mean corpuscular hemoglobin concentration; Mean corpuscular hemoglobin; Reticulocyte count (absolute); Platelet count; White blood cell count; Neutrophil count (absolute); Lymphocyte count (absolute); Monocyte count (absolute); Eosinophil count (absolute); Basophil count (absolute); Large unstained cells.
Coagulation Parameters: Activated partial thromboplastin time; Fibrinogen; Prothrombin time
Clinical Chemistry Parameters: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Creatine Kinase, Total bilirubina, Urea nitrogen, Creatinine, Calcium, Phosphorus, Total protein, Albumin, Globulin (calculated), Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride.
Thyroid Sample Collection: P Generation On the day of scheduled euthanasia, blood samples were collected from all P generation male and female rats. F1 Generation Pups: On Day 3 postpartum, blood samples were collected from 2 culled pups/litter/group. On Day 12 postpartum, blood samples were collected from 1 pup/sex/litter/group.
Thyroid Sample Analysis: Serum samples were analyzed for Thyroxine (T4) levels using a validated analytical procedure.

Ovarian and Uterine Examinations
The reproductive tract was dissected from the abdominal cavity. The number of implantation sites was recorded.

Necropsy
All P generation males and females were subjected to a complete necropsy examination.

Organ Weights
The organs were weighed at necropsy for all P generation rats at scheduled euthanasia. Paired organs were weighed together, unless otherwise indicated.

Tissue Collection and Preservation
Representative samples of the tissues were collected from all rats and preserved in 10% neutral buffered formalin.

Histology
Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

Histopathology
Histopathological evaluation was performed by a board-certified veterinary pathologist. The following tissues were evaluated:
• Thyroid and parathyroid were evaluated from all P generation males and females in the control and high dose groups.
• Gross lesions were evaluated in all P generation males and females in all groups.
• Tissues identified were evaluated from all P generation males and females in the control and high dose groups.
• Special attention was paid to the stages of spermatogenesis in the testes, epididymides, and interstitial testicular cell structures.
• Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated, processed, and reported.
Thyroid and parathyroid were evaluated from one F1 generation pup/sex/litter in the control and high dose groups

STATISTICAL ANALYSIS
Descriptive statistics including number, mean, percentages and/or standard deviation were reported as appropriate.
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the study


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations:once weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / isoflurane/oxygen anesthesia
- Animals fasted: Yes
- How many animals:5/sex/group
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- How many animals: :5/sex/group
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine:5/sex/group
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:all groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Functional Observation Battery, Motor Activity.

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during cohabitation, until evidence of spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period. On the day of scheduled euthanasia (LD 14), an examination of vaginal cytology was performed prior to necropsy examination to determine the stage of estrous cycle.
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by serial vaginal lavage procedures.
Sperm parameters (parental animals):
Stage dependent qualitative evaluation of spermatogenesis in the testis, epididymides, and interstitial testicular cell structures was performed.
Litter observations:
- Litter Size (defined as all pups delivered);
- Litters were observed for dead pups at least twice daily;
- F1 generation pups had body weights recorded on Day 0 (birth), 3, 6, 9, and 12 postpartum;
- Anogenital distance was recorded for all F1 generation pups using a calibrated stereomicroscope;
- Nipple presence was evaluated and the number present (if any) was recorded for all F1 generation male pups;

Postmortem examinations (parental animals):
- Clinical Pathology;
- Coagulation;
- Clinical Chemistry;
- Hematology;
- Thyroid Sample Analysis and Evaluation;
- Ovarian and Uterine Examinations;
- Necropsy;


Necropsy
Postmortem examinations (offspring):
- Thyroid hormone (T4 assessment);
- Thyroid and parathyroid gland (histology);
- Organ Weights;
- Tissue Collection and Preservation
Statistics:
Descriptive statistics including number, mean, percentages and/or standard deviation were reported as appropriate. Litter values were used where appropriate. Additional procedures and/or analyses may be performed, if appropriate. Clinical and necropsy observations data were summarized but no inferential statistical analysis were performed. Statistically significant pair-wise comparison probabilities were reported as either p ≤ 0.05 or p ≤ 0.01, unless otherwise noted below.
Reproductive indices:
- Duration of Gestation: The duration of gestation was calculated from GD 0 to the day the first pup is observed;
- Fertility Index: Percentage of matings that result in pregnancies. ( Number of Animals Pregnant x 100)/ (Number of Animals Mated);
- Mating Index: Percentage of animals in cohabitation that mated. (Number of Animals Mated x 100)/ Number of Animals in Cohabitation
- Pregnancy Rate: Percentage of animals in cohabitation that result in pregnancies. (Number of Animals Pregnant x 100)/ (Number of Animals in Cohabitation)
- Gestation Index: Percentage of pregnancies that result in birth of live litters. (Number of Animals with Live Offspring x 100)/(Number of Animals Pregnant)
- Number of offspring per litter: Live and dead pups;
- Number of implantation sites;
- General condition of dam and litter during the postpartum period
Offspring viability indices:
- Viability Index: Percentage of pups born that survive 4 days postpartum. (Number of Live Pups on Day 4 Postpartum x 100)/(Number of Liveborn Pups on Day 1 Postpartum);
- Lactation Index: Percentage of pups that survive 134 days postpartum. (Number of Live Pups on Day 134 Postpartum x 100)/ (Number of Live Pups on Day 4 Postpartum)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All P generation males survived to scheduled euthanasia, and there were no Bourgeonal-related clinical signs in the P generation males at any dose. Mean body weights, mean body weight gains, and mean food consumption values were similar across all groups in the P generation males. There were no Bourgeonal-related effects on any neurobehavioral parameter (functional observation battery or motor activity) at any dose. There were no Bourgeonal-related effects on any mating and fertility parameter in the P generation males at any dose. There were no Bourgeonal-related macroscopic or microscopic observations or alterations in organ weights at any dose. In addition, there were no Bourgeonal related effects on hematology, clinical chemistry, or coagulation parameters in the P generation males at any dose. There were no Bourgeonal-related changes in serum T4 concentrations in the P generation males at any dose. In the P generation males, mean serum T4 concentrations were 104%, 87%, and 90% of controls in the 0.5, 1, and 5 mg/kg/dose groups, respectively, on DS 43/44. This effect was not dose-dependent and not associated with any macroscopic or microscopic observations or alteration in thyroid weight.
Dermal irritation (if dermal study):
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no Bourgeonal-related mortality in the P generation females at any dose.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All differences in hematology parameters were considered unrelated to Bourgeonal because: 1) the observations were not dose dependent; 2) the differences were of small magnitude; and/or 3) the differences were inconsistent in direction.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no Bourgeonal-related effects on clinical chemistry parameters in the P generation males and females at any dose.
All differences in clinical chemistry parameters, including those that reached statistical significance, were considered unrelated to Bourgeonal because: 1) the observations were not dose dependent; 2) the differences were of small magnitude; and/or 3) the differences were inconsistent in direction.

There were no Bourgeonal-related changes in serum T4 concentrations in the P generation males at any dose. In the P generation males, mean serum T4 concentrations were 104%, 87%, and 90% of controls in the 0.5, 1, and 5 mg/kg/dose groups, respectively, on DS 43/44. This effect was not dose-dependent and not associated with any macroscopic or microscopic observations or alteration in thyroid weight.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no Bourgeonal-related effects on any neurobehavioral parameter (functional observation battery or motor activity) at any dose.
There were no Bourgeonal-related effects on ambulation or fine movement during motor activity testing in the P generation males at any dose.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The test substance, Bourgeonal, and vehicle control formulations were administered to male and female Crl:CD(SD) Sprague-Dawley rats at doses of 0, 0.5, 1, or 5 mg/kg/dose by once daily oral gavage 7 days a week for a minimum of 28 days. Parental (P generation) male rats were administered the test or control substance formulations once daily via oral gavage beginning 14 days before cohabitation with treated females, during cohabitation and continuing through the day prior to scheduled euthanasia on Days 43 through 46. P generation males were exposed to the test or control substance for a minimum of 28 days. P generation female rats were administered the test or control substance formulations once daily via oral gavage beginning 14 days before cohabitation with treated males and continuing through Lactation Day 12 (rats that delivered a litter) or Gestation Day 24 (rats that did not deliver a litter).
F1 generation pups were not directly exposed to the test or control substance, but may have been possibly exposed during maternal gestation (in utero exposure), via maternal milk during the lactation period, or from exposure to maternal urine/feces.
In the P generation, female No. 1407 in the 1 mg/kg/dose group was found dead on Day 39, and female No. 1434 in the 0.5 mg/kg/dose group was found dead on Day 45. The causes of death were not evident microscopically and considered undetermined and unrelated to the test substance.

There were no microscopic changes attributed to Bourgeonal in the 5 mg/kg/dose P generation males or females, or in the thyroid glands or testes/epididymides of any groups.

No microscopic changes in the P generation animals were attributed to the test substance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no Bourgeonal-related effects on mating and fertility. The days in cohabitation (2.8 to 3.5 days), mating index (90% or 100%), and fertility index (90% or 100%) in the 0.5, 1, and 5 mg/kg/dose groups were similar to the control group values.
Pregnancy occurred in 9 (90%), 10 (100%), 9 (90%), and 10 (100%) of the 10 mated females in the 0, 0.5, 1, and 5 mg/kg/dose groups, respectively. Of these pregnant females, 8 to 10 females across the groups delivered their litters and one dam in the 1 mg/kg/dose group was found dead
on GD 22, as previously described. There were no Bourgeonal-related effects on any natural delivery or litter parameter at any dose. The mean number of implantation sites per delivered litter, dams with stillborn pups, dams with no liveborn pups, gestation index (number of rats with live offspring/number of pregnant rats), mean number of dams with all pups dying (Days 0 to 3 postpartum and Days 4 to 12 postpartum), mean number of pups delivered (liveborn and stillborn), pups found dead or presumed cannibalized, percent male pups per number of pups sexed per litter, surviving pups per litter, lactation index, and live litter size were similar among the four dose groups.
There was a statistically significant decrease (p≤0.01) in the number of pups found dead between Days 1 and 3 postpartum at 0.5 and 1 mg/kg/dose resulting from an increase in pup mortality in the control group during this same period. Consequently, the viability index in the 0.5 and 1 mg/kg/dose groups was higher (p≤0.01) than the control group.

Details on results (P0)

There were no Bourgeonal-related effects on mating and fertility. The days in cohabitation (2.8 to 3.5 days), mating index (90% or 100%), and fertility index (90% or 100%) in the 0.5, 1, and 5 mg/kg/dose groups were similar to the control group values. There were no Bourgeonal-related effects on gross pathology, body weight gain, food consuption, organ weight.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
General Systemic Toxicity
Effect level:
>= 5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive performance
Remarks on result:
other:
Remarks:
no test substance related adverse effect findings

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
5 mg/kg bw/day (nominal)

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no Bourgeonal-related clinical signs observed in the F1 generation pups at any dose.
Dermal irritation (if dermal study):
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no Bourgeonal-related effects on mean body weights in the F1 generation pups at 0.5 and 1 mg/kg/dose. The mean pup body weight was statistically significantly reduced (p≤0.05 or p≤0.01) in the 5 mg/kg/dose group compared to control values on Days 9 and 12 postpartum (88% to 89% of controls). The reduced pup weights were considered unrelated to the Bourgeonal because they were within the range observed historically at the Testing Facility.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no Bourgeonal-related changes in serum T4 concentrations in the F1 generation males or females at any dose. In the F1 generation male pups, mean serum T4 concentrations were 98%, 82%, and 78% of controls in the 0.5, 1, and 5 mg/kg/dose groups, respectively, on Day 12 postpartum. In the F1 generation female pups, mean serum T4 concentrations were 82%, 74%, and 74% of controls in the 0.5, 1, and 5 mg/kg/dose groups, respectively, on Day 12 postpartum. There were no Bourgeonal-related microscopic changes in the thyroid or parathyroid glands of the single F1 generation pup/sex/litter that was microscopically examined from 5 mg/kg/dose group. The differences observed in mean serum T4 concentrations in the F1 generation females did not reflect any other evidence at the tissue level, therefore, were considered unrelated to administration of Bourgeonal.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There were no Bourgeonal-related differences in mean anogenital distance in the F1 generation males or females at any dose on Day 1 postpartum.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no Bourgeonal-related differences on nipple retention in the F1 generation male pups in any dose group.
No male pups had nipples present on PND 12.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no Bourgeonal-related macroscopic findings noted in the F1 generation males or females at any dose. The macroscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of Sprague-Dawley rats and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of Bourgeonal.
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no Bourgeonal-related microscopic findings in the thyroid gland or parathyroid gland in the single F1 generation pup/sex/litter that was microscopically examined from the 5 mg/kg/dose group.
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

There were no Bourgeonal-related clinical signs observed in the F1 generation pups at any dose. There were no Bourgeonal-related effects on mean body weights in the F1 generation pups at 0.5 and 1 mg/kg/dose. There were no Bourgeonal-related changes in serum T4 concentrations in the F1 generation males or females at any dose. There were no Bourgeonal-related differences on nipple retention in the F1 generation male pups in any dose group. There were no Bourgeonal-related macroscopic findings noted in the F1 generation males or females at any dose. The macroscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of Sprague-Dawley rats and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of Bourgeonal. There were no Bourgeonal-related microscopic findings in the thyroid gland or parathyroid gland in the single F1 generation pup/sex/litter that was microscopically examined from the 5 mg/kg/dose group.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
Neonatal
Generation:
F1
Effect level:
>= 5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test substance related adverse effect findings
Remarks on result:
other: no test substance related adverse effect findings

Overall reproductive toxicity

Reproductive effects observed:
no
Lowest effective dose / conc.:
5 mg/kg bw/day (nominal)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, Bourgeonal was administered via oral gavage to male and female Crl:CD(SD) Sprague Dawley rats once daily beginning before cohabitation, through mating and continuing for at least 28 days (P generationmale rats;actual; 42 to 45 doses) or through parturition until Day 12of lactation (P generation femalerats;actual:38 to 56 doses) at doses of 0.5, 1, or 5 mg/kg/dose. Administration of Bourgeonal did not produce any mortality or clinical signs in the P generation males or females at any dose. There were no Bourgeonal-related effects on mating and fertility in the P generation males or females or any effects on estrous cycling and natural delivery parameters in the P generation females. There were no Bourgeonal-related differences in any preweaning developmental parameter evaluated in the F1 generation offspring. Furthermore, there were no Bourgeonal-related macroscopic or microscopicfindings in the P generation adults or F1 generation pupsor alterations in the organ weights in the P generation adults. Based on these findings, the no-observed-adverse-effect-level (NOAEL) for general toxicity, mating, and fertility for Bourgeonal in P generation males and females was 5 mg/kg/dose. The NOAEL for development of the F1 generation offspring was also 5 mg/kg/dose.
Executive summary:

The objectives of this study were to detect potential effects that may result from daily exposure of Bourgeonal to Crl:CD(SD) male and female rats beginning before cohabitation, through mating and continuing for at least 28 days (male rats) or through parturition until Day 12of lactation (female rats).

The study design was as follows:

Text Table1
Experimental Design

Group No.

Test Material

Dose Level (mg/kg/dose)

Concentration
(mg/mL)

Dose Volume (mL/kg/dose)

No. of Rats

Males

Females

1

Control

0

0

15

10

10

2

Bourgeonal

0.5

0.033

15

10

10

3

Bourgeonal

1

0.067

15

10

10

4

Bourgeonal

5

0.33

15

10

10

 

A total of 80 naïve Crl:CD(SD) Sprague Dawley P generation rats (10 rats/sex) were randomly assigned to four dosegroups(Groups 1 through 4), 10 rats/sex/group. Formulations of the test substance, Bourgeonal, or the control substance, corn oil, were administered orally by gavage once daily beginning before cohabitation, through mating and continuing for at least 28 days (P generation male rats;actual: 42 to 45 doses) or through parturition until Day 12of lactation (P generation female rats; actual: 38 to 56 doses)at 0 (Control), 0.5, 1, and 5 mg/kg/dose.

The following parameters and end points were evaluated in the P generation rats assigned in this study: viability, clinical observations, detailed clinical signs, body weights and body weight changes, food consumption, neurobehavioral evaluations (functional observation battery and motor activity), mating and fertility assessments, organ weights, and macroscopic and microscopic evaluations. P generation females were further evaluated for estrous cyclicity, natural delivery parameters, and maternal observations.

The following parameters and end points were evaluated in the F1 generation pups: viability, clinical signs, pup body weights and internal sex, preweaning parameters (anogenital distance and nipple retention), and macroscopic and microscopic examinations.

Blood samples were collected from all P generation males and females for clinical pathology assessments (hematology, coagulation,andclinical chemistry) and from P generation males and F1 generation pups for thyroid hormone assessments.

P Generation Males

All P generation males survived to scheduled euthanasia, and there were no Bourgeonal‑related clinical signs in the P generation males at any dose. Mean body weights, mean body weight gains, and mean food consumption values were similar across all groups in the P generation males. There were no Bourgeonal-related effects on any neurobehavioral parameter (functional observation battery or motor activity) at any dose.

There were no Bourgeonal-related effects on any mating and fertility parameter in the P generation males at any dose. There were no Bourgeonal-related macroscopic or microscopic observations or alterations in organ weights at any dose. In addition, there were no Bourgeonal-related effects on hematology, clinical chemistry, or coagulation parameters in the P generation males at any dose.

There were no Bourgeonal-related changes in serum T4 concentrations in the P generation males at any dose. In the P generation males, mean serum T4 concentrations were 104%, 86%, and 90% of controls in the 0.5, 1, and 5 mg/kg/dose groups, respectively, on DS 43/44. This effect was not dose dependent and not associated with any macroscopic or microscopic observations or alteration in thyroid weight.

P Generation Females

There were no Bourgeonal-related mortality in the P generation females at any dose. A total of1 and 1P generation females in the0.5 and 1 mg/kg/dose groups, respectively, did not survive to scheduled euthanasia. Of these unscheduled necropsies, two P generation females were found dead prior to or after dose administration. These unscheduled necropsies were considered unrelated to Bourgeonalbecause the deaths were not dose-dependent. In-life and postmortem observations in these rats are summarized below. All remaining P generation female rats survived to scheduled euthanasia

There were no Bourgeonal-related clinical observations or effects on mean body weights, mean body weight gains, and mean food consumption values during the premating, gestation, or lactation periods in the P generation females at any dose. There were no Bourgeonal-related effects on any neurobehavioral parameter (functional observation battery or motor activity) at any dose.

There were no Bourgeonal-related effects on estrous cycling, mating or fertility, or any natural delivery or litter parameter in the P generation females at any dose. There were no Bourgeonal‑related macroscopic or microscopic observations or alterations in organ weights at any dose.

In P generation females, there were no Bourgeonal-related effects on any hematology, coagulation, or clinicalchemistryparameter at any dose.

F1 Generation Pups

In the F1 generation pups, there were no Bourgeonal-related clinical observations or effects on anogenital distance, nipple retention (males), or mean pup body weights at any dose. In addition, there were no Bourgeonal-related macroscopic or microscopic observations in the F1 generation pups at any dose.

There were no Bourgeonal-related changes in serum T4 concentrations in the F1 generation males or females at any dose. In the F1 generation male pups, mean serum T4 concentrations were 98%, 82%, and 78% of controls in the 0.5, 1, and 5 mg/kg/dose groups, respectively, on Day 12postpartum. Inthe F1 generation female pups, mean serum T4 concentrations were 82%, 74%, and 74% of controls in the 0.5, 1, and 5 mg/kg/dose groups, respectively, on Day 12postpartum. There were no Bourgeonal-related microscopic changes in the thyroid or parathyroid glands of the single F1 generation pup/sex/litter that was microscopically examined from 5 mg/kg/dose group. The differences observed in mean serum T4 concentrations in the F1 generation females did not reflect any other evidence at the tissue level, therefore, were considered unrelated to administration of Bourgeonal.

In conclusion, Bourgeonal was administered via oral gavage to male and female Crl:CD(SD) Sprague Dawley rats once daily beginning before cohabitation, through mating and continuing for at least 28 days (P generationmale rats;actual; 42 to 45 doses) or through parturition until Day 12of lactation (P generation femalerats;actual:38 to 56 doses) at doses of 0.5, 1, or 5 mg/kg/dose. Administration of Bourgeonal did not produce any mortality or clinical signs in the P generation males or females at any dose. There were no Bourgeonal-related effects on mating and fertility in the P generation males or females or any effects on estrous cycling and natural delivery parameters in the P generation females. There were no Bourgeonal-related differences in any preweaning developmental parameter evaluated in the F1 generation offspring. Furthermore, there were no Bourgeonal-related macroscopic or microscopicfindings in the P generation adults or F1 generation pupsor alterations in the organ weights in the P generation adults. Based on these findings, the no-observed-adverse-effect-level (NOAEL) for general toxicity, mating, and fertility for Bourgeonal in P generation males and females was 5 mg/kg/dose. The NOAEL for development of the F1 generation offspring was also 5 mg/kg/dose.